Antibiotics that inhibit cell growth by interfering with protein synthesis have been among the most clinically successful antibacterials. In spite of the importance of these inhibitors, there are significant gaps in our understanding of the most fundamental principles of their action. Many of the protein synthesis inhibitors, from the classic chloramphenicol (CHL) to the newer linezolid (LZD), bind at the catalytic peptidyl transferase center (PTC) of the ribosome, where they clash with the placement of aminoacyl-tRNA. Because of the location of their binding site, it is commonly assumed that they inhibit translation by interfering with formation of every peptide bond, either at the start codon or at any of the internal codons of a gene. However, our preliminary data show that this view is principally incorrect. Instead of indiscriminately inhibiting peptide bond formation, CHL and LZD stall elongation of translation only at specific mRNA sites. The nature of the nascent peptide chain appears to play the major role in specifying the sites of translation arrest, but the general rules that define the sites of stalling and the molecular mechanisms that underlie this effect remain unknown. Therefore, the main goal of this project is to gain a detailed understanding of the context specific action of PTC-targeting antibiotics. The study will primarily focus on LZD and CHL. LZD is the first and most broadly medically used oxazolidinone. CHL is one of the oldest known ribosomal antibiotics. In spite of its reduced medical importance, inclusion of CHL in the study is crucial, not only because of the vast amount of information available for this inhibitor, but also to contrast its context specific action with that of LZD and correlate the effects with individual structural properties of the drugs. In Specific Aim 1, whole-cell ribosome profiling and quantitative biochemical testing will be used to identify the detailed requirements for the sequence context that defines the preferred sites of inhibition of translation by LZD or CHL. In Specific Aim 2, an array of biochemical, structural and genetic approaches will be employed to understand the molecular mechanisms that account for the context-specific action of the PTC- targeting inhibitors. The use of innovative techniques, such as single molecule FRET or an engineered tethered ribosome, are expected to provide principally new insights into the most fundamental aspects of action of the inhibitors of the ribosomal catalytic center. Specific Aim 3 will address a conceptually important and medically-relevant question, whether context specificity of drug action results into protein-specific inhibition of translation by the PTC-targeting antibiotics. The anticipated findings should significantly expand the understanding of the general mode of action of clinically-important antibacterials that act upon the catalytic center of the ribosome and may open new venues for rational development of protein synthesis inhibitors with superior antibiotic properties.